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J. Biol. Chem., Vol. 276, Issue 33, 31376-31387, August 17, 2001
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§,§,§,§,§,§, and¶
From the Department of Molecular and Developmental
Biology, Institute of Medical Science, The University of Tokyo, Tokyo
108-8639, § CREST, Japan Science and Technology Corp. (JST),
Tokyo 108-8639, and ¶ Tokyo Metropolitan Institute of
Medical Science, Tokyo 113-8613, Japan
Received for publication, March 12, 2001, and in revised form, June 8, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Dfp1/Him1 protein of fission yeast,
Schizosaccharomyces pombe, encodes the regulatory subunit
for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for
initiation and progression of the S phase of the cell cycle. This
protein binds and activates Hsk1 kinase, which phosphorylates the MCM2
protein. Comparison of the amino acid sequences of the Cdc7 regulatory
subunits from various eukaryotes revealed the presence of three small
stretches of conserved amino acid sequences, namely Dbf4 motifs N, M,
and C. We report here that the Dbf4 motif M, a unique proline-rich motif, and the Dbf4 motif C, a C2H2-type zinc
finger motif, are essential for mitotic functions of Dfp1/Him1 protein
as well as for full-level activation of Hsk1 kinase. In
vitro, a small segment containing the Dbf4 motif M or C alone
binds to and partially activates Hsk1. Co-expression of these two
segments augments the extent of activation. Furthermore, a fused
polypeptide containing only Dbf4 motifs M and C without any spacer can
activate Hsk1 and is capable of rescuing the growth defect of
him1 null cells. Insertion of a long stretch of amino acids
between the motif M and motif C can be tolerated for mitotic functions.
On the other hand, internal deletion of Dbf4 motif N, which has some
similarity with the BRCA C-terminal domain motif, results in a
defect in hydroxyurea-induced checkpoint responses and sensitivity to
methyl methane sulfonate, yet mitotic functions and kinase activation are intact. In one-hybrid assays with budding yeast Dbf4, motif N
mutants exhibit reduced interaction with a replication origin. Our
observations suggest the molecular architecture of Cdc7·Dbf4-related kinase complexes at the origins, in which they are tethered to replication machinery through Dbf4 motif N and the catalytic subunits are activated through bipartite binding of Dbf4 motifs M and C of the
regulatory subunits.
Saccharomyces cerevisiae CDC7 is a
temperature-sensitive mutant defective in initiation of DNA replication
(1). Initial experiments indicated that ongoing protein synthesis was
not required for completion of the S phase once the function of Cdc7
was executed (2, 3). dbf4 (dumbbell
former), isolated from independent screening (4), showed
terminal phenotypes similar to those of cdc7(ts). Later,
DBF4 was re-isolated as a multi-copy suppressor of
cdc7(ts) (5). Subsequent genetic and biochemical evidence showed that DBF4 encodes a regulatory or activation subunit
for Cdc7 protein (6).
Structural and functional homologues of Cdc7 have been noted in fission
yeast, human, mouse, and Xenopus (7-10). The kinase domains
are particularly well conserved and identity between yeast and human is
45%. Activation subunits for these Cdc7-related kinases have been
isolated through interaction screening or in data base searches
(11-17). Expression of the activation subunit is cell cycle-regulated
and is accumulated during the S phase (13, 17- 21). Accordingly,
Cdc7-dependent kinase activity is high during the S phase.
Despite functional similarity to cyclins in apparent periodic
appearance and kinase activation during the cell cycle, Dbf4 and
cyclins share no apparent sequence similarity. Compared with the Cdc7
catalytic subunits, Dbf4 and activation subunits for Cdc7-related
kinases from other eukaryotes are more diverged. There is less than
25% identity between Dbf4 and Dfp1/Him1, the fission yeast homologue
of Dbf4, and no overall homology was evident between yeast and
mammalian Dbf4 homologues. However, alignment of known Dbf4 homologues
revealed two stretches of amino acids (Dbf4 motif N and Dbf4 motif C)
conserved in all the known Dbf4/Him1-related molecules (13). We
discovered another stretch of amino acids (Dbf4 motif M),which is also
conserved in all the Dbf4-related molecules (22).
With fission yeast Dfp1/Him1 protein as a model, we have
now generated a series of deletion and point mutants and examined the
functions of these conserved motifs of Dbf4-related molecules regarding
in vivo functions, binding to the catalytic subunit, kinase
activation, and interaction with replication origins. We describe here
essential functions of Dbf4 motif M and Dbf4 motif C for mitotic cell
cycle and kinase activation and the potential of each motif to serve as
an independent Hsk1 binding module. The combination of these two motifs
can activate the kinase activity of Hsk1. We also found that Dbf4 motif
N, related to BRCA C-terminal domain motif although not
essential for mitotic function and kinase activation, plays crucial
roles in the DNA replication checkpoint as well as in recovery from DNA
damage-induced cell cycle arrest and may be involved in interaction
with replication machinery or chromatin.
Yeast Strains, Media, and
Genetics--
Schizosaccharomyces pombe strains were grown
in rich (YES) or minimal
(EMM)1 medium containing the
required supplements. General genetic manipulation (23) and
transformation (24) were done as described. To induce expression from
the nmt1 or modified nmt1 promoter (25), cells were grown to
mid-exponential phase in EMM containing 10 µg/ml thiamine, spun down,
and washed three times with EMM lacking thiamine before being
resuspended in fresh medium lacking thiamine. Cell-survival analysis
for DNA replication block or DNA damages was done as described (26).
Construction of Deletion Derivatives of Dfp1/Him1--
Deletion
derivatives were PCR-constructed using sets of primers listed on Table
I. For PCR, we used Taq
DNA polymerase Hi-Fi (Roche Molecular Biochemicals) as outlined
by the supplier. The amplified fragment, digested with SalI
and BamHI, was inserted into the pREP41-HA vector (25). For
expression in insect cells, the resulting expression vector on
pREP41-HA was first digested with NdeI, filled in with the
Klenow fragment, and digested with BamHI. The insert DNA
containing the HA tag and him1+-coding frame was
re-cloned at the SmaI-BglII site of pVL1393, an
insect cell expression vector.
Construction of Internal Deletion and Point
Mutants--
Internal deletions and point mutations were introduced
during two consecutive runs of PCR. The first PCR was done using sets of N1/antisense primers and BmR/sense primers (Table I). The resulting
two fragments were isolated and used for the second PCR in the presence
of N1 and BmR primers. The resulting fragment was subcloned into
pREP41-HA and pVL1393 vectors as described above, and the presence of
the expected mutations was confirmed by sequencing. The construction of
motif N mutants of Dbf4 was done on pGAD424-Dbf4 fusion plasmid (27) as
were with two sets of primers (Gal4AD-sense/antisense and
sense/Gal4AD-antisense), and the final PCR products were digested with
EcoRI and PstI and re-cloned into pGAD424.
Construction of Motif M-Motif C Fusion and Expression of Two
Domains on One Plasmid--
Dbf4 motif C (61 amino acids) was
PCR-amplified with motif C-Bam(N) and motif C-BglII(C), and
the resulting fragment was cloned at the BamHI site of
pREP41-motif M followed by fill-in of the XhoI site present
just upstream of the BamHI site. The resulting plasmid
expressed 113 amino acids of Dbf4 motif M (223) fused to 61 amino
acids of Dbf4 motif C (485) through a 5-amino acid linker
sequence. To construct a plasmid expressing Dbf4 motif M and Dbf4 motif
C, the PstI-SmaI fragment of pREP41-motif C
containing nmt1-driven Dbf4 motif C was cloned at the SmaI
site of pREP41-motif M using a PstI-SmaI adapter.
The resulting plasmid simultaneously expressed HA-tagged 113-amino acid
motif M and 65-amino acid motif C polypeptides.
Preparation of Extracts from Insect Cells, Immunoprecipitation,
and in Vitro Kinase Assays--
Expression of proteins in insect cells
and preparation of extracts were done as previously described (17).
Immunoprecipitation and kinase assays were also done as described (10,
17).
Antibodies--
Anti-HA monoclonal antibody (12CA5) was
purchased from Berkeley Antibody Co. Rabbit anti-Hsk1 antibodies pep1
(9) and Hsk1C were developed against an oligopeptide (corresponding to
residues 361-378 of Hsk1 protein) and bacterially produced GST-Hsk1C
(residue 423-506), respectively.
One-hybrid Assays--
One-hybrid assays were done with JYL363
(ura3::{URA3
Gal Complementation Assays and Effect of HU and MMS on S/M Checkpoint
and Survival--
Plasmids expressing wild-type or mutant forms of
him1+ were transformed into
him1+/ The Hsk1 Kinase Attenuated Mutant Hsk1K129R-K130S Could Complement
hsk1(ts) and Be Activated by Dfp1/Him1--
Hsk1 is a serine-threonine
kinase, the kinase activity of which is essential for cell cycle
progression of fission yeast cells (9). We generated three mutant forms
of Hsk1, Hsk1K129D, Hsk1K129N, and Hsk1K129R-K130S, in which the
conserved lysine at position 129 or at both 129 and 130 were replaced
with aspartic acid, asparagine, or arginine-serine, respectively. The
lysine at position 129 is conserved in serine-threonine kinases (42).
The pREP1-based plasmid carrying Hsk1K129D, Hsk1K129N, or
Hsk1K129R-K130S under the inducible nmt1 promoter was introduced into a
novel hsk1(ts) mutant, hsk1-89, which could not
grow at temperatures above 30 °C because the kinase activity was
impaired (43). We examined the potential of the three Hsk1
mutants to rescue the growth of hsk1-89 by examining the
growth of each strain at 30 °C. Although Hsk1K129D and Hsk1K129N
could not rescue the growth of hsk1-89, hsk1-89
carrying Hsk1K129R-K130S could grow at the nonpermissive temperature,
albeit with a slightly reduced rate with no thiamine in the media (Fig.
1A). At 25 °C, all the
transformants grew, although Hsk1K129D and Hsk1K129N exhibited a
dominant-negative growth inhibition probably due to titration of
Dfp1/Him1 protein by inactive kinases (Fig. 1A). Therefore,
Hsk1K129D and Hsk1K129N are probably inactive kinases, whereas
Hsk1K129R-K130S may retain some kinase activity.
For confirmation, we expressed these three mutant Hsk1 in insect cells
and examined their kinase activities in the presence and absence of
Dfp1/Him1 protein. All the mutants could form complexes with Dfp1/Him1,
as indicated by the co-immunoprecipitation of Him1 by the anti-Hsk1
antibody (Fig. 1B, lanes 1-4). Activation of
Hsk1 kinase was assessed by phosphorylation of associated Dfp1/Him1 protein derivatives as well as by phosphorylation of GST-SpMCM2N containing the N-terminal 220 amino acids of fission yeast Cdc19/MCM2 protein. Upon phosphorylation by an active Hsk1·Dfp1/Him1 kinase complex, a mobility-shifted form of GST-SpMCM2N, which migrated more
slowly on SDS-PAGE, was detected (Fig. 1C, lanes
5 and 11). Although the non-shifted form of GST-SpMCM2N
is also phosphorylated, the majority of this phosphorylation appears to
be caused by a nonspecific kinase present in immunoprecipitates.
Therefore, we will evaluate the extent of Hsk1 kinase activation by
examining the appearance of this mobility-shifted form of the
substrate. Both Hsk1K129D and Hsk1K129N showed no kinase activity (Fig.
1C, lanes 2 and 3), and
Hsk1K129R-K130S was only slightly active in autophosphorylation and was
inactive in phosphorylation of MCM2 in the absence of Him1 protein
(Fig. 1C, lane 4). However, when Dfp1/Him1 was
co-expressed, Hsk1K129R-K130S was activated, and both
autophosphorylation of Hsk1 and efficient phosphorylation of MCM2
protein were evident (Fig. 1C, lanes 8 and
12). In contrast, two other mutants were not activated, even
in the presence of Dfp1/Him1; hence, these findings are consistent with
the notion that these mutants are inactive (Fig. 1C,
lanes 6, 7, 9, and 10). Dfp1/Him1 was hyperphosphorylated, as indicated by appearance of a
smear of mobility-shifted forms on SDS-PAGE when complexed with the
wild-type Hsk1, through kinase activity of the latter protein (Fig.
1C, lanes 5 and 11). However,
Hsk1K129R-K130S in complex with Dfp1/Him1, although capable of
phosphorylating itself and MCM2, did not induce hyperphosphorylation of
Dfp1/Him1 (Fig. 1C, lanes 8 and 12).
These results indicate that Hsk1K129R-K130S cannot be fully activated
by Dfp1/Him1 or that the mutation caused a conformational change of the
complex that led to inadequate recognition of phosphorylation sites on
the associated Dfp1/Him1 protein.
Three Stretches of Amino Acids Conserved in Cdc7 Regulatory
Subunits--
Despite the striking functional conservation of
regulatory subunits for Cdc7-related kinases, the level of conservation
of their primary structure is low. There is only a 25% overall
identity between budding yeast Dbf4 and fission yeast Dfp1/Him1, and no significant homology was identified between ASK, a regulatory subunit for huCdc7, and Dbf4, except for two small stretches of amino
acids (Dbf4 motif N and Dbf4 motif C), which are also conserved in
Dfp1/Him1. We earlier identified another stretch of amino acids, Dbf4
motif M, conserved among all the known Dbf4-related molecules (Ref. 22;
Fig. 2A). These motifs are
conserved not only in known Cdc7 regulatory subunits but also in Spo6,
another Dbf4-related molecule in fission yeast, the function of which
is specific for sporulation (29).
Complementation Activity of Deletion Derivatives of
Dfp1/Him1--
To better understand functions of each conserved domain
in Dfp1/Him1 protein, we generated a series of N-terminal and
C-terminal deletion derivatives. In generating deletions, we made use
of the information on the exon-intron organization of
spo6+ (29). We selected end points of deletions
at positions of introns, since exon junctions often define the
structural boundaries (30). The truncated proteins were expressed on
plasmid pREP41 under the control of the attenuated nmt1 promoter. The
mitotic function of each deletion, defined as the potential to rescue
the growth of him1 null cells when expressed on a plasmid,
was examined by introducing them into the
him1+/ Regions of Dfp1/Him1 Required for Binding and Activation of
Hsk1--
We then expressed each deletion derivative in insect cells
to determine interactions with and activation of Hsk1 protein (Fig. 3). Each mutant Dfp1/Him1 was
co-expressed either with wild-type Hsk1 or the kinase-attenuated mutant
of Hsk1 (K129R-K130S) to calibrate the extent of activation of the
kinase by the mutant proteins. The Dfp1/Him1 deletions were tagged with
HA at the N terminus, and immunoprecipitation was done either with
anti-HA or anti-Hsk1 antibody. Binding to Hsk1 was evaluated by
co-immunoprecipitation of the two proteins with either antibody. Kinase
assays were done on the immunoprecipitates with GST-SpMCM2N used as a
substrate, as described in the previous section. We first confirmed
that the expression of Hsk1 and each deletion derivative of Dfp1/Him1 was at a similar level in insect cell extracts (data not shown). Consistent with the result of mitotic functions, Del5 (104) bound to Hsk1 and activated its kinase activity as efficiently as did the
wild type. Del6 (177) and Del7 (223) also bound to and
activated both Hsk1 wild type and K129R-K130S, as indicated by their
potential to cause a mobility shift of GST-SpMCM2N protein, although
Del7 was only partially active. Therefore, Dbf 4-motif N (151) may also contribute to kinase activation and/or recognition of the substrate. Alternatively, conformational change induced by the deletion
may be inhibitory for functions.
On the other hand, the C-terminal deletion, which profoundly affected
the mitotic function of Him1, also affected its kinase activation. Del4
(1) and Del11 (1), lacking the C-terminal 34 and 65 amino
acids, respectively, could activate the wild-type Hsk1 but not the
attenuated Hsk1K129R-K130S. Hsk1K129R-K130S -Del4 or
Hsk1K129R-K130S-Del11 combination generated very little phosphorylated and mobility-shifted forms of GST-SpMCM2N, whereas full-level phosphorylation of MCM2 was observed in combination with the wild-type Hsk1. This was the case even with Del3 (1). Del8 (336) and
Del10 (223) also similarly activated the wild-type Hsk1 but not
K129R-K130S, although the level of MCM2 phosphorylation was reduced
compared with former deletions containing the N-terminal regions. This is consistent with findings that deletion of the N-terminal segment of
Dfp1/Him1, although not essential for mitotic functions, decreased its
potential to activate Hsk1 for phosphorylation of MCM2. The level of
kinase activation was particularly affected in immunoprecipitates with
anti-HA antibody, which suggests that the presence of the HA antibody
may be inhibitory for kinase activation and/or recognition of the
substrate in the N-terminal deletions.
The results indicate that both N-terminal and
C-terminal regions can independently interact with Hsk1 and activate
kinase activity in vitro. Del1 (1), Del2 (1), and
Del12 (336), which lack both Dbf4 motif M and C, showed very
little or no binding and activation of Hsk1. However, Del10 (223),
carrying the Dbf4 motif M, was capable of activating the wild-type Hsk1
and underwent extensive phosphorylation. In conclusion, activation of
Hsk1 kinase, as revealed by the potential to activate the K129R-K130S
mutant, required at minimum the C-terminal 323 amino acids, containing both Dbf4 motif M and Dbf4 motif C, which are also sufficient to
maintain its mitotic function. However, either the N-terminal 335 amino
acids or the C-terminal 210 amino acids containing Dbf4 motif M or Dbf4
motif C, respectively, were sufficient for binding and activation of
the wild-type Hsk1 for MCM2 phosphorylation. Binding and activation was
completely lost in the absence of both motifs.
A Mutation in Dbf4 Motif M Results in Impaired Mitotic Functions
and Kinase Activation--
Our findings suggest that both Dbf4 motif M
and Dbf4 motif C play crucial roles in kinase activation and mitotic
function of Dfp1/Him1 protein. To determine whether Dbf4 motif M plays essential roles on full-length Dfp1/Him1 protein, we generated a mutant
Dfp1/Him1 in which conserved residues in this motif (two aromatic
residues, Tyr-311 and Tyr-291, and one aspartic acid, Asp-286) were
replaced with alanine (Fig. 2A). The mutant protein, expressed on the pREP81 vector under the highly attenuated nmt1 promoter, was able to rescue growth of the him1 null cells
on plates without thiamine, although the growth of the transformants was slower than seen in the wild-type cells (Fig.
4A). The levels of the
Dfp1/Him1 protein in transformants in the absence of thiamine were
similar between the wild-type and the mutant (Fig. 4B).
After the addition of thiamine to the culture grown in medium lacking thiamine, growth of the motif M mutant strain was almost completely suppressed, whereas the strain carrying pREP81-Him1 (wild type) grew
after the addition of thiamine (Fig. 4A).
Fluorescence-activated cell sorter analyses of DNA content in
asynchronous culture of the motif M mutant after the addition of
thiamine showed an increase in 1C cells (Fig. 4C,
right; 3 h) followed by an increase of less than
1C DNA content (Fig. 4C, right; 6-24
h). These results indicate that the motif M mutant cells first
arrest at the G1/S boundary, then proceed to premature
mitosis in the absence of DNA synthesis. A significant portion of the
cells exhibited the cut phenotype, indicative of problems in coupling
of DNA synthesis and mitosis (data not shown).
The kinase activation by the motif M mutant was then examined in
vitro. The motif M mutant bound to Hsk1 with an efficiency similar
to that seen in the wild-type (data not shown) but failed to fully
activate its kinase activity. The mobility-shifted form of MCM2 was not
detected in combination with the K129R-K130S mutant, and the extent of
the Him1 hyperphosphorylation was also significantly reduced (Fig.
4D, lanes 5, 6, 11, and
12). These results indicate that the motif M mutant is
defective in full-level activation of Hsk1 kinase.
Bipartite Hsk1 Binding Modules on Dfp1/Him1 Protein--
The
results of deletion and mutation analyses indicate that the presence of
either Dbf4 motif M or Dbf4 motif C may be sufficient for binding to
Hsk1 and for partial kinase activation in vitro. We then
asked if small segments containing Dbf4 motif M or Dbf4 motif C would
be sufficient for binding to Hsk1. The 113-amino acid segment from
amino acids 223 to 335 (Fig. 2B, Motif M) or the
65-amino acid segment from amino acids 481 to 545 (Fig. 2B; Motif C) was co-expressed with Hsk1 in insect cells. The
mutant Dbf4 motif M containing three amino acid substitutions
(Motif M mutant) was also similarly expressed. Either
polypeptide containing the Dbf4 motif M or the Dbf4 motif C was
co-immunoprecipitated with Hsk1 protein (Fig.
5A, lanes 1,
2, 7, and 8; data not shown), thus
indicating that both motifs are capable of independently binding to
Hsk1. The mutant Dbf4 motif M polypeptide could not bind to Hsk1 (Fig.
5A, lanes 3, 4, 9, and
10), suggesting that the mitotic defect of the motif M
mutant was due to its reduced efficiency of interaction with Hsk1,
although this effect could not be detected on the full-length Dfp1/Him1
protein in our immunoprecipitation assay. Kinase assays with the
immunoprecipitate containing motif M or motif C complexed with Hsk1
indicated that both the wild-type Hsk1 and associated motif M or motif
C polypeptide could be autophosphorylated (Fig. 5B,
lanes 1 and 5). However, the level of
autophosphorylation of Hsk1K129R-K130S was lower either with motif M or
motif C compared with the full-length Dfp1/Him1 protein (Fig.
5B, lanes 2, 6, and 8).
Similarly, very little MCM2 phosphorylation (appearance of a
mobility-shifted form) was detected with either polypeptide in a
complex with wild-type Hsk1 (Fig. 5B, lanes 1 and
5), indicating that additional segments of Dfp1/Him1 are
required for kinase activation. We further narrowed down the two
binding regions by constructing plasmids expressing 86-amino acid
(250) and 60-amino acid (276) segments containing motif M and
58-amino acid (488) and 37 amino acid (488) segments
containing motif C (Fig. 5C). Immunoprecipitation with
anti-HA antibody followed by detection with anti-Hsk1 antibody plus
kinase assays indicated that the 86- and 60-amino acid motif M and
58-amino acid motif C were able to associate with Hsk1 (Fig.
5D, lanes 3-8), but the 37-amino acid motif C
was not (Fig. 5D, lanes 9 and 10).
Autophosphorylation of the wild-type Hsk1was detected with the 86-amino
acid motif M and 58-amino acid motif C and with the 60-amino acid motif
M to a lesser extent (Fig. 5D, lanes 3,
5, and 7). Phosphorylation of the 86-amino acid
motif M in a complex with the wild-type Hsk1 was also evident (data not
shown). Although the 86-amino acid motif M and 58-amino acid motif C
supported autophosphorylation of Hsk1K129R-K130S to a significant
extent (Fig. 5D, lanes 4 and 8), the
60-amino acid motif M, which bound to Hsk1, showed the level of Hsk1
K129R-K130S autophosphorylation at about 20% that shown by the
86-amino acid motif M polypeptide (Fig. 5D, lane 6). These results define the minimal segment required for Hsk1 binding within the 60- and 58-amino acid segments of motif M and motif
C, respectively, and suggest that the 60-amino acid motif M segment,
capable of binding to Hsk1, lacks amino acids essential for full-level
activation of Hsk1 kinase.
We recently found that the co-expression of the N-terminal and
C-terminal halves of ASK, the activation subunit for huCdc7, together
with huCdc7 in mammalian cells could reconstitute an active
kinase.2 Therefore, we
expressed both motif M and motif C polypeptides together with Hsk1
protein in insect cells. When the immunoprecipitates were assayed for
kinase reactions, more efficient phosphorylation of both polypeptides
(as revealed by more extensive mobility shift) was observed with
wild-type Hsk1 protein (Fig. 5B, lanes 11 and 15). The level of Hsk1K129R-K130S phosphorylation also
increased (Fig. 5B, lanes 12 and 16).
This stimulation was not observed when the motif M mutant was
coexpressed with motif C (Fig. 5B, lanes 13,
14, 17, and 18). These results suggest
a possibility that the motif M and motif C polypeptides form a ternary
complex with Hsk1, which then may lead to stimulation of Hsk1 kinase
activity. It should be noted that MCM2 phosphorylation was not
stimulated even in the presence of both motif M and motif C (Fig.
5B, compare lanes 1, 2, 5,
6, 15, and 16).
Only Motif M and Motif C Are Sufficient for Mitotic Activity and
Kinase Activation--
The above results suggest the possibility that
only motif M and motif C segments are sufficient for mitotic activity
of Dfp1/Him1 protein and that the segment connecting the two motifs may
be dispensable. The amino acid sequences between Dbf4 motif M and Dbf4
motif C are not conserved between species, and the length also varies,
with 170, 346, and 37 amino acids in Dfp1/Him1, Dbf4, and ASK,
respectively. To determine if these sequences contribute to mitotic
activity and kinase activation of Dfp1/Him1 protein, we constructed a
plasmid expressing a fused polypeptide composed of only small segments
of motif M and motif C. The resulting polypeptide contains 113 amino
acids (223) of motif M, 5 amino acids derived from an unrelated
linker sequence and 61 amino acids (485) of motif C. Surprisingly,
this small polypeptide could support growth of him1 null
cells (Fig. 6A,
construct b). This result clearly indicates that the
intervening sequences between motif C and motif M are not required for
mitotic activity and kinase activation of Dfp1/Him1 protein and that
the presence of the two conserved motifs is sufficient. We then
determined if insertion of an exogenous sequence between the two motifs
could be tolerated. We found that the presence of a 37- or 346-amino
acid sequence derived from the intervening sequence of human ASK or
budding yeast Dbf4, respectively, did not abrogate mitotic activity
(Fig. 6A, constructs c and d). The M-C
fusion polypeptide expressed in insect cells could bind and activate
Hsk1 kinase activity (Fig. 6B, lanes 3,
4, 8, and 9), although the level of
MCM2 phosphorylation was much lower than that seen in the presence of
full-length Dfp1/Him1 protein. These results suggest that amino acid
sequences between the two motifs serve only as a flexible spacer
segment that connects two essential segments. Expression of two
separate motif M and motif C polypeptides was not capable of
complementing the him1 null cell (Fig. 6A,
construct e); hence, the simultaneous presence of the two
segments as a fused protein is needed for mitotic function.
Role of Motif N in Control of DNA Replication Checkpoint--
The
above results indicate that only Dbf4 motif M and Dbf4 motif C are
required for mitotic functions of Dfp1/Him1 and that Dbf4 motif N is
dispensable. We reported that mutations in the BRCA C-terminal
domain-like Dbf4 motif N (3A mutant in which conserved serine/threonine
residues, Ser-183, Thr-187, and Thr-191, were substituted with alanine
and Him1 Dbf4 Motif N Is Required for Interaction with Replication Origins
in Vivo--
Dowell et al. (31) report that a one-hybrid
assay could detect the interaction of S. cerevisiae Dbf4
protein with replication origins in a sequence-specific manner. The
segment on Dbf4 involved in this interaction was mapped to positions
81-278, which contained the motif N. Therefore, we asked if the
BRCA C-terminal domain-like motif N is involved in the
interaction of Dbf4 protein with replication origins. We constructed
Dbf4 mutants It is now well established that Cdc7-related kinases are widely
conserved and play pivotal roles in initiation of eukaryotic DNA
replication (14, 32-35). Although the catalytic subunits encoded by
Cdc7-related genes share significant homology in kinase-conserved domains, structures of the regulatory subunits are unexpectedly diverged (13). Through comparison of Dbf4, Dfp1/Him1, and ASK, the
regulatory subunits from budding yeast, fission yeast, and human,
respectively, we identified and reported three stretches of amino acids
that are conserved in all the three molecules (13, 17, 22). In the
present work, using fission yeast Dfp1/Him1 protein as a model, we
carried out detailed analyses of structures involved in various
functions of this essential regulator of the Cdc7 kinase, the ultimate
switch for DNA replication.
Dissection of Dfp1/Him1 Protein; Hsk1 Binding, Kinase Activation,
and Mitotic Function--
Brown and Kelly (11) report that Dfp1/Him1
changes the substrate specificity rather than activating Hsk1 kinase
activity per se. Hsk1 expressed in insect cells possesses
significant autophosphorylation activity on its own and to some extent
can phosphorylate exogenous substrates. The Hsk1K129R-K130S mutant we
generated shows very little autophosphorylation activity by itself,
which means that its intrinsic kinase activity is significantly
impaired. However, its autophosphorylation as well as MCM2
phosphorylation activities can be activated almost to the wild-type
level in the presence of Dfp1/Him1 protein. Use of the Hsk1K129R-K130S
mutant facilitated the measurement of the potential of various mutant
Dfp1/Him1 proteins to activate the kinase reactions mediated by Hsk1.
The Dfp1/Him1 mutants we characterized can be classified
into eight types depending on the conserved motifs they carry (Fig. 8). The type C or D, lacking or mutated
in either Dbf4 motif C or M, respectively, lost mitotic activity
completely or partially, and this correlates with their attenuated
kinase activation, as revealed by their lack of potential to activate
attenuated Hsk1 mutant Hsk1K129R-K130S. Truncation mutants of nimO
protein, the Aspergillus homologue of Dbf4, lacking the
motif C segment was reported to be incapable of complementing the
nimO deletion (12). Similarly, C-terminal truncation of
budding yeast Dbf4 was shown to lose complementation activity of a
dbf4 null mutant (5). Both type C and D can bind to Hsk1 and
activate the wild-type Hsk1 in vitro, and type F or G,
containing only Dbf4 motif M or C, respectively, can bind to Hsk1.
However, Hsk1 binding and, thus, kinase activation is completely lost
in type E lacking both Dbf4 motif M and C. In contrast, Dbf4 motif N is
dispensable for mitotic functions and full-level kinase activation; the
C-terminal 323 amino acids are sufficient for complementing growth
defect of him1 null cells. However, type B mutants lacking
or mutated in Dbf4 motif N are partially defective in DNA replication
checkpoint control and are sensitive to DNA damages.
Our results indicate that Dfp1/Him1 protein interacts with
Hsk1 through two separate binding domains, Dbf4 motif M and C. Dbf4
motif C is related to C2H2-type zinc finger
motif (12), which is often involved in protein-protein interactions
(36), whereas Dbf4 motif M, characterized by the presence of conserved multiple proline residues, is not related to any known motifs in the
data base and, hence, may be a novel protein-interacting motif. Our
earlier two-hybrid analyses showed that the C-terminal 50 amino acids
containing Dbf4 motif C could interact with Hsk1, findings consistent
with data obtained in the immunoprecipitation studies. In each complex
containing the 113-amino acid motif M or 65-amino acid motif C, the
Dfp1/Him1-derived polypeptide was phosphorylated. However, MCM2
phosphorylation was not stimulated in either case. This indicates that
additional segments are required for activation of kinase for
phosphorylation of exogenous substrates. Del3 (1), lacking Dbf4
motif C but containing motif N and motif M, could activate MCM2
phosphorylation on the wild-type Hsk1 (Fig. 3). In contrast, Del8
(336) or Del10 (223), containing either Dbf4 motif C or Dbf4
motif M but lacking the N-terminal region, showed a reduced level of
MCM2 phosphorylation, especially with anti-HA immunoprecipitates. The
level of MCM2 phosphorylation, in complex with either wild-type or
K129R-K130S mutant, was lower with Del7 (223) compared with Del5
(104), although both deletions contained Dbf4 motif M and Dbf4
motif C (Fig. 3). These results indicate that segments between 104 and
222 may contribute to kinase activation. Alternatively, the presence of
an additional segment N-terminal to Dbf4 motif M may facilitate
efficient recognition of the substrate protein.
Coexpression of motif M and motif C polypeptides of Hsk1 in insect
cells resulted in enhanced autophosphorylation of Hsk1K129R-K130S as
well as that of motif M and motif C polypeptides but not
phosphorylation of MCM2 (Fig. 5B, lanes 11-18).
Although we cannot conclude from these experiments that the three
molecules form a ternary complex, it is possible that simultaneous
binding of motif M and motif C to distinct regions on Hsk1 may
synergistically stimulate its kinase activity, whereas interaction with
exogenous substrates may require an additional segment of Dfp1/Him1 protein.
The presence of two Hsk1 binding regions is consistent with data by
Hardy and Pautz (37) that two separate regions on Dbf4 protein can bind
to Cdc7 in two-hybrid assays. Furthermore, the two Dbf4 regions mapped
in the above studies, 241-416 and 573-695 of Dbf4, contain Dbf4 motif
M and Dbf4 motif C, respectively. Furthermore, the mutation in the
temperature-sensitive dbf4-1, dbf4-2, or
dbf4-3 was identified, respectively, as a proline to leucine substitution at position 277 and a proline to leucine or serine
substitution at position 308 within Dbf4 motif M (Ref. 38; Fig.
2A). These two proline residues are conserved in Dbf4 motif
M. Furthermore, nimO18 mutation was identified to be a
substitution of a rather conserved valine residue within Dbf4 motif M
with glutamic acid (12). These findings support our conclusion that Dbf4 motif M is crucial for functions of Cdc7 regulatory subunits.
Dbf4 Motif N Is Required for Replication Checkpoint Functions and
Is Involved in Interaction with Chromatin--
Our results also
indicate that domains on Dfp1/Him1 protein essential for mitotic
functions and those involved in replication checkpoint functions could
be separated. Sensitivity to HU observed with some of our mutants may
not be simply due to additive effects of HU on S phase defects. The
motif N mutants are sensitive to HU and develop cut cells in the
presence of HU despite their proficiency for kinase activation and
mitotic functions. The sequence similarity of motif N to the
BRCA C-terminal domain-like domain on Cut5 protein (17) suggests
the presence of a common factor that may interact with this domain. The
amino acids 81-278 of Dbf4 protein were previously noted to be
sufficient for interaction with ARS in one-hybrid assays (31). This
region contains the entire Dbf4 motif N. Our results with one-hybrid
assays showed that Dbf4 motif N is required for interaction with
replication origins in vivo. It is an intriguing possibility
that motif N interacts with replication machinery assembled at the
origin, and this interaction, although dispensable for mitotic
functions, may be important for signal transmission of DNA replication
checkpoint control. Identification of interacting molecules with the
motif N will aid in identifying molecular architecture involving
Dbf4-related molecules at the origins.
Mode of Cdc7 Kinase Activation by Cdc7 Regulatory
Subunits--
It was concluded that kinase activation to a full extent
requires the presence of both Dbf4 motif M and C. Since these two motifs are not related in amino acid sequences, it is likely that the
two domains bind to distinct domains on Hsk1 protein. The kinase
activities of budding yeast Cdc7 and human Cdc7 strictly depend on
association with corresponding regulatory subunits. Given the high
degree of amino acid conservation of these motifs, we predict that
activation of Cdc7-related kinase by Dbf4-related regulatory subunits
may commonly involve these two conserved domains. A segment of ASK, the
activator of huCdc7, containing either motif M or motif C, can
independently interact with huCdc7, and a segment containing both Dbf4
motif M and Dbf4 motif C of ASK is sufficient for activation of huCdc7
in vivo and in vitro. The coexpression of two
independent N-terminal and C-terminal huCdc7 binding segments of ASK
with huCdc7 resulted in activation of autophosphorylation.2
Although both 60-amino acid (276) and 86-amino acid (249)
Dbf4 motif M segments bound to Hsk1 with similar efficiency, a higher level of kinase activation was observed with the latter polypeptide. This suggests that the 27 amino acids (249) are required for
kinase activation.
LX3KX6RD (254),
present immediately N-terminal to Dbf4 motif M, is conserved in budding
yeast Dbf4 and ASK and may play important roles in activation of Hsk1 kinase.
The sequences and lengths between motif M and motif C are
diverged even between budding and fission yeasts. Polypeptides
containing only Dbf4 motif M and C but lacking the spacer sequence
rescued the growth defect of him1 null cells. Furthermore,
insertion of 37-amino acid or 346-amino acid spacer sequences derived
from ASK or Dbf4, respectively, between the motif M and motif C on the
same plasmid did not affect complementing activity. Thus, our results
demonstrate that simultaneous binding of motif M and motif C segments
to the catalytic subunit is sufficient for kinase activation as well as
for mitotic functions and that the intervening region serves merely as
a flexible spacer peptide that connects the two conserved motifs for
coordinated binding to the catalytic subunit (Fig. 8). This is in
contrast to activation of Cdk by cyclins, where all the Cdk binding
activity is contained in a single domain of ~100 amino acids in
length called the cyclin box (39). A unique feature of the Cdc7 kinase
family is the presence of two large insert sequences that interrupt the
kinase domains. The presence of these kinase inserts is essential for kinase activity of Cdc7
(40).3 It is an intriguing
issue as to how Dbf4 motif M and Dbf4 motif C interacts with this
unique class of protein kinases and how this interaction leads to
generation of an active kinase. It was reported that budding yeast Cdc7
is in an oligomeric form through self-interactions (41). Furthermore,
oligomerization of Dbf4 and nimO protein was suggested, based on the
ability of C-terminal-truncated proteins to complement
temperature-sensitive mutants (12). Thus, Cdc7 and its regulatory
subunit may generate a complex containing multiple molecules of each
protein through more complex protein-protein interactions.
Delineation of the domains on Dfp1/Him1, a cyclin-like molecule
essential for DNA replication, provides an important clue for future
study on precise mechanisms of kinase activation, interaction with
replication machinery at the origins, and the molecular basis of the
role in control of DNA replication checkpoint. Furthermore, identification of small peptides that can bind and modulate functions of this essential kinase will provide a basis for developing novel strategies by which cell cycle progression of human cells can be
manipulated through modulation of activities of the huCdc7-ASK kinase complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides used for construction of plasmids
) and
YJL365 (ura3::{URA3
Gal) as
previously reported (28). For the LacZ assay in liquid culture, we used
yeast 
-galactosidase assay kits (Pierce).
heterozygous diploid cells. The
transformants were sporulated, and him1 cells
carrying the plasmid were screened after random spore formation. Growth
of him1 null cells was monitored on EMM plates containing thiamine and adenine as well as in liquid culture to determine the
efficiency of complementation. To examine the effect of HU on
checkpoint control, vegetatively growing transformants or mutant strains (5 × 106 cells/ml) were grown in the presence
of 10 mM HU, 10 µg/ml thiamine, and 10 mM
adenine. Aliquots were taken every 2 h to count the number of
"cut" cells using fluorescence microscopy. To determine the MMS
sensitivity of him1 null cells carrying him1
mutants, growing cells (5 × 106 cells/ml) in EMM in
the presence of adenine and thiamine were treated with 0.05% MMS for
3 h, then 1000 cells were plated on minimal plates. After
incubation for 5 days, colony formation was examined to determine cell
survival and recovery of growth.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The mitotic function and kinase activity of
Hsk1 mutants. A, complementation of
temperature-sensitive growth of hsk1-89 by overexpression
of Hsk1 wild-type (WT) and its mutants. The
hsk1-89 cells carrying the plasmid indicated were spread on
EMM plates lacking thiamine. Plates were incubated at 25 °C for 1 week or at 30 °C (non-permissive for hsk1-89) for 5 days. B, Hsk1, wild-type, or mutant, as shown (lanes
1-4), was coexpressed with HA-tagged Dfp1/Him1 protein in insect
cells, and extracts were prepared. In lanes 5, only
HA-tagged Dfp1/Him1 protein was expressed. Immunoprecipitates
(IP) with anti-Hsk1 (Hsk1C) antibody (upper
panel) or the untreated extracts (lower panel) were
analyzed by Western blotting on SDS-PAGE with anti-HA antibody.
Dfp1/Him1 complexed with the wild-type Hsk1 was mobility-shifted on
SDS-PAGE and appears as multiple bands due to phosphorylation, whereas
that complexed with a mutant Hsk1 protein is not. Dfp1/Him1 complexed
with Hsk1K129R-K130S can be slightly mobility-shifted depending on the
running condition. C, Hsk1, wild-type or mutant as shown,
was expressed alone (lanes 1-4) or was coexpressed with
HA-tagged Dfp1/Him1 (lanes 5-12) in insect cells.
Immunoprecipitates prepared either by anti-Hsk1 antibody (Hsk1C;
lanes 1-8) or by anti-HA antibody (lanes 9-12)
were incubated in the case of kinase reactions in the presence of
GST-SpMCM2N protein as a substrate, and the products were analyzed on
8% SDS-PAGE, followed by autoradiography. * and # indicate
mobility-shifted (phosphorylated) and non-shifted forms of the
substrate, respectively.
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Fig. 2.
Alignment of the three conserved motifs of
Dbf4-related molecules and Dfp1/Him1 mutants characterized in this
study. A, locations of the characterized three
conserved motifs in Dfp1/Him1 protein are indicated as striped
regions. The dark gray portion indicates the region
that shares 25% identity with S. cerevisiae Dbf4, whereas the light gray portions represent less
conserved regions. The sequence alignments of the three motifs between
Dbf4, Dfp1/Him1, and huASK are shown in addition to the positions of
point mutations in Dbf4 motif N and Dbf4 motif M mutants generated.
Amino acid substitutions at the conserved proline residues of Dbf4
motif M in dbf4-1, -2, and -3 temperature-sensitive mutants (38) as well as those at conserved valine
residues in nimO18 mutant (12) are also indicated. The amino
acid residues conserved in more than two members are indicated by
white letters in black backgrounds. aa, amino
acids. B, schematic drawing of Dfp1/Him1 mutants
constructed and analyzed in this study. The gray and
striped regions represent the N-terminal HA tag and the
three motifs, respectively. The numbers in parentheses
indicate the amino acid residues present in each deletion derivative.
For small motif M and motif C polypeptides, the lengths of amino acids
are also indicated. The potential of each derivative, when expressed on
the pREP41 vector in the presence of thiamine, to rescue the growth of
the him1 null mutant is indicated for each mutant. +++ and
++ indicate full growth recovery and slower cell growth. + indicates
that the mutant can complement only in the absence of thiamine, where
the protein is overexpressed. Him1 FL, full-length
Him1.
heterozygous diploid cells (Fig.
2B). The N-terminal deletion up to the amino acid 222 (Del
7) did not significantly affect the potential to complement the growth
defect of the him1 disruptant. Further deletion up to the
position 335, which removed Dbf4 motif M (Del 8), resulted in loss of
complementation activity. On the other hand, deletion of the C-terminal
35 amino acids, removing a part of Dbf4 motif C (Del 4), led to reduced
complementing activity; the mutant protein restored the growth only in
medium lacking thiamine, in which nmt1 promoter was activated. Further
deletion up to position 480, which removed the entire Dbf4 motif C
(Del11), led to a complete loss of complementation activity. Thus,
C-terminal 323 amino acids are sufficient for mitotic function of
Dfp1/Him1 protein, and both Dbf4 motif M and C may be essential for
this function.
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Fig. 3.
Immunoprecipitation (IP) and
kinase assays with Dfp1/Him1 deletion derivatives expressed in insect
cells. A, Hsk1, either wild-type (lane 1 and
even-numbered lanes) or kinase-attenuated K129R-K130S
(odd-numbered lanes other than lane 1) was
co-expressed with HA-tagged Dfp1/Him1 mutants in insect cells, and
extracts were prepared. Some of the Dfp1/Him1 derivatives are
extensively phosphorylated when complexed with the wild-type Hsk1 and
appear as multiple mobility-shifted bands. The arrowheads
indicate the positions of the non-shifted Dfp1/Him1 derivatives, as
observed in a complex with Hsk1K129R-K130S. Immunoprecipitation was
done either by anti-Hsk1 (Hsk1C) antibody (A) or anti-HA
antibody (B). The immunoprecipitates were run on SDS-PAGE
and then blotted (IB) to detect the proteins indicated
(upper and middle panels; anti-Hsk1 pep1 antibody
and anti-HA antibody, respectively). They were also incubated in kinase
reactions in the presence of GST-SpMCM2N protein as a substrate
(lower panels). For blots with anti-Hsk1 antibody and kinase
assays, samples were run on 8% SDS-PAGE, whereas they were run on 10%
(lanes 1-17 in A and lanes 1-15 in
B) or 11% (lanes 18-27 in A) or 13%
(lanes 16-27 in B) SDS-PAGE for anti-HA blot. On
11 and 13% SDS-PAGE, the gels were run under conditions in which
separation of phosphorylated forms would be minimized; therefore, the
mobility shifts are less obvious than for other gels. * and # indicate
mobility-shifted and non-shifted forms of the substrate, respectively.
IgG indicates immunoglobulin heavy chain. FL,
full-length.
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Fig. 4.
Defect in mitotic function and kinase
activation of a motif M mutant. A-C, him1
null cells carrying pREP81-HAHim1 (motif M mutant) or
pREP81-HAHim1 (wild-type (WT)) were grown in EMM without
thiamine. At time 0, thiamine was added to 10 µg/ml to half of the
culture and was kept growing at 30 °C. For the motif M mutant, two
independent clones (clones 1 and 2) were examined. A,
A600 was measured at the times indicated
after the addition of thiamine. B, extracts were prepared
from him1 null cells carrying various plasmids and were
analyzed by Western blotting. Lanes 1 and 2,
pREP81 vector; lanes 3 and 4, pREP81-HAHim1 (wild
type (WT)); lanes 5 and 6,
pREP81-HAHim1 (motif M mutant), clone 1; lane 7,
pREP81-Him1 (no tag); lane 8, insect cell extract expressing
HA-tagged Him1 protein. Lanes 1, 3, 5,
and 7, grown in the absence of thiamine; lanes 2,
4, and 6, grown in the presence of thiamine.
Upper, blot (IB) with anti-HA antibody;
lower, blot with anti-tubulin antibody. C, DNA
contents of the cells at the times indicated after the addition of
thiamine were measured by fluorescence-activated cell sorter analyses.
Left column, wild-type; right column, motif M
mutant clone 1. D, Hsk1, either wild-type (odd
numbered lanes) or K129R-K130S mutant (even-numbered
lanes) was coexpressed with HA-tagged Dfp1/Him1 mutants, as
indicated, in insect cells, and extracts were prepared.
Immunoprecipitation (IP) was done either with an anti-HA
antibody (lanes 1-6) or by anti-Hsk1 (Hsk1C) antibody
(lanes 7-12). Immunoprecipitates were incubated in kinase
reactions in the presence of GST-SpMCM2N protein as a substrate. * and
# indicate mobility-shifted and non-shifted forms of the substrate,
respectively.
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Fig. 5.
Immunoprecipitation (IP) and
kinase assays with motif M or motif C polypeptides. In
A, B, and D, Hsk1, either wild-type
(odd-numbered lanes) or K129R-K130S mutant
(even-numbered lanes), was co-expressed with HA-tagged
full-length Dfp1/Him1 protein (FL), a Dfp1/Him1 mutant,
motif M (Mwt and Mmut, derived
from the wild-type and mutant, respectively), or motif C segment
(C) as indicated in insect cells, and extracts were
prepared. The lengths of the motif M and motif C segments where not
indicated are 113 and 65 amino acids, respectively. A,
immunoprecipitates by anti-Hsk1 (Hsk1C) antibody (lanes
1-6) or by anti-HA antibody (lanes 7-12) were run on
8% SDS-PAGE and were blotted (IB) by anti-Hsk1 pep1
antibody (upper panel) or run on 15% SDS-PAGE and were
blotted by anti-HA antibody (lower panel). The upper
band in lanes 7-12 of anti-Hsk1 blot is a
phosphorylated form of Hsk1, which occasionally appears on SDS-PAGE
depending on the running condition. B, kinase assays were
done using immunoprecipitates prepared with anti-HA antibody
(lanes 1-10, 15-18) or with anti-Hsk1 (Hsk1C)
antibody (lanes 11-14) in the presence of GST-SpMCM2N
protein, and products were analyzed on an 8% SDS-PAGE (upper
panel) or on a 15% SDS-PAGE (lower panel).
aa, amino acids. C, Dbf4 motif M or C
polypeptides tagged with HA epitope at the N terminus with indicated
lengths were expressed in insect cells. Extracts were run on a 15%
SDS-PAGE and blotted using the HA antibody. D,
immunoprecipitates by anti-HA antibody were used for in
vitro kinase assays in the presence of GST-SpMCM2N protein, and
products were analyzed on a 8% SDS-PAGE (upper panel). The
broad band above Hsk1 is phosphorylated and mobility-shifted Dfp1/Him1
protein. The same kinase reaction mixtures were analyzed by Western
blotting using anti-Hsk1 (pep1) antibody (lower panel). In
B and D, * and # indicate mobility-shifted and
non-shifted forms of the substrate, respectively. FL,
full-length.
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Fig. 6.
Only the Dbf4 motif M and Dbf4 motif C
regions are sufficient for mitotic functions of Dfp1/Him1, and the
intervening region can be deleted or replaced with other sequences of
different lengths. A, schematic drawing of the plasmids
expressing fusion polypeptides of motif M and motif C stretches
containing various intervening sequences and their properties. The
level of growth complementation is expressed as in Fig. 2B.
++ or + in the Hsk1 activation in vitro column indicates
phosphorylation of exogenous MCM2 substrate or activation of only
autophosphorylation but not that of substrate phosphorylation,
respectively. ND, not determined. aa, amino
acids. B, in vitro kinase assays with a motif M
and motif C fusion polypeptide (containing a 5-amino acid spacer;
construct b shown in A) expressed in insect cells.
Combinations of Hsk1, wild-type (WT), or mutant as indicated
and Dfp1/Him1 (full-length (FL) or M-C fusion
(M-C) were expressed in insect cells, and in
vitro kinase assays were done on immunoprecipitates
(IP) prepared by anti-HA (lanes 1-5) or by
anti-Hsk1 (Hsk1C) (lanes 6-10) antibody. The products were
run on 8% (upper and lower) or 15%
(middle) SDS-PAGE. Upper panel, autoradiogram of
kinase assay; middle panel, blot (IB) with
anti-HA antibody; lower panel, blot with anti-Hsk1 (pep1)
antibody. IgG indicates immunoglobulin heavy and light
chains.
154-193 lacking the entire motif N; see Fig. 2A)
led to a checkpoint defect after exposure to HU as well as growth
retardation in recovery from DNA damages (17). We generated additional
mutants of this motif and examined their properties (Fig.
7A). Him1177-222 lacks the
C-terminal half of the motif N, including conserved serine/threonine
residues. Him1154-222 lacks the entire motif N as well as
additional amino acids, whereas Him1-3E contains serine/threonine
(Ser-183, Thr-187, and Thr-191) to glutamic acid substitutions. All the
above mutants except for Him1-3E could support a near normal growth in
him1 null cells when expressed from the attenuated nmt1
promoter on pREP41 (Fig. 2B), and the levels of kinase
activation were comparable with those of the wild-type (data not
shown). However, all the mutants exhibited mild sensitivity to HU, and
cut cells were generated after exposure to HU to the level about
50-60% that of the cds1 mutant (Fig. 7,
A and B). The Cds1 protein is known to be
required for S-phase checkpoint control downstream of Rad3/ATM kinase. Him1 3A, Him1 3E, Del6, and Del 7 were also sensitive to MMS. These
results further confirm our previous observation and strongly support
our conclusion that Dbf4 motif N is specifically involved in response
of cells to HU-mediated replication fork block as well as in proper
recovery from DNA damage-induced cell cycle arrest.
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Fig. 7.
HU and DNA damage sensitivity of
him1 null cells carrying various motif N mutants and
origin binding activity of budding yeast Dbf4 motif N mutants.
A, schematic drawing of Dfp1/Him1 mutants lacking all or
part of Dbf4 motif N and their properties in S/M checkpoint and MMS
sensitivity. MMS-sensitive (S) strains showed less than 30%
survival after treatment with 0.05% MMS and showed markedly reduced
cell growth even after a 5-day incubation period. The wild-type strain
showed 50% survival and full growth recovery under the same conditions
(indicated as R). ND, not determined.
B, him1 null cells carrying
him1+ (open squares),
him1-3A (closed squares),
him1 177-222 (closed circles),
him1154-222 (open circles), and
cds1 cells (open triangles) were
grown in EMM, and HU was added to 10 mM at time 0. Cut
cells were counted at the times indicated, and their fractions in total
population are expressed with each mutant. C, one-hybrid
assays with motif N mutants of budding yeast Dbf4. Bait plasmids were
introduced into YJL363 (with wild-type ARS consensus sequence
(WT-ACS), columns 1-5) or into YJL365 (with mutant ARS
consensus sequence, column 6), and LacZ activity was measured.
Column 1, pGAD424 vector; columns 2 and
6, pGAD424-Dbf4 (wild-type (WT)); column
3, pGAD424-Dbf4 (N-2E); column 4,
pGAD424-Dbf4 (N-GL); column 5, pGAD424-Dbf4
(N). The values represent the averages of the measurement of six
independent clones. FL, full-length.
N, GL, and 2E, in which the N-terminal half of Dbf4
motif N (residues 126-160) is deleted or conserved LG at position
158-159 was changed to GL or the conserved threonine residues at
positions 171 and 175 were substituted for by glutamic acid,
respectively. These mutants were expressed as fusions with the
activation domain of the Gal4 transcription factor on plasmid pGAD424.
The resulting plasmids rescued the temperature-sensitive growth of
dbf4-1(ts) mutant (data not shown). These results are consistent with our conclusion that Dbf4 motif N is not required for
mitotic functions and interaction with the catalytic subunit in
Dfp1/Him1. To determine if these motif N mutants could interact with
replication origins in one-hybrid assays, wild-type Dbf4 for the
control could interact with wild-type ARS sequences but not with
mutant ARS-containing base substitutions within the ACS (ARS consensus
sequence), indicating that the assay indeed measures the
sequence-specific interaction of Dbf4 protein with replication origins.
In contrast to the wild type, all three motif N mutant Dbf4 plasmids
showed a background level of LacZ activity (Fig. 7C), thus
indicating loss of specific interaction with replication origins.
Therefore, Dbf4 motif N is specifically required for interaction with
replication machinery or chromatin components at the origins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Summary of functional domains of Dfp1/Him1
and a possible mode of Hsk1 kinase activation at origins.
A, summary of the requirement of conserved domains of
Dfp1/Him1 protein for mitotic function, Hsk1 binding, and kinase
activation. Types A-H represent classes of Dfp1/Him1
mutants classified on the basis of the presence (+) and absence ( 
) of
each conserved motif or each activity. +/ in the types F
and G indicate that kinase activation may depend on the
presence of additional sequences outside the conserved Dbf4 motif M. B, schematic drawing of architecture of Dfp1/Him1 protein on
the chromatin. Dfp1/Him1 protein interacts with Hsk1 through two
conserved modules, Dbf4 motif M and Dbf4 motif C, shown as
circles marked with M and C, respectively. The
sequences between them, represented by wavy lines, are not
conserved in other Dbf4-related molecules and are not required for
interaction with and activation of Hsk1. They constitute a flexible
spacer segment that connects the two motifs. Dfp1/Him1 protein may bind
to different portions of the Hsk1 through the bipartite independent
binding modules, and this will lead to conformational change of the
catalytic subunit to activate its kinase activity. Motif N, not
essential for mitotic function when the protein is provided on a
plasmid, may interact with replication machinery on the chromatin, thus
facilitating phosphorylation of replication components by Hsk1. See
"Discussion" for details. WT, wild
type.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Akio Sugino and Joachim Li for providing unpublished results on mutation sites of dbf4 mutants and for generous gifts of plasmids and strains for Dbf4 one-hybrid assays, respectively. We also thank Noriko Sato for information on experiments on reconstitution of active huCdc7 kinase complex from two truncated ASK molecules, members of our laboratory for helpful discussion, and M. Ohara for helpful comments and for language assistance.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell
Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; E-mail: hmasai@rinshoken.or.jp.
Published, JBC Papers in Press, June 11, 2001, DOI 10.1074/jbc.M102197200
2 N. Sato, M. Sato, K. Arai, and H. Masai, unpublished data.
3 H. Masai, E. Matsui, K. Ogino, and K. Arai, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: EMM, Edinburgh minimal medium; PCR, polymerase chain reaction; HA, hemagglutinin; GST, glutathione S-transferase; HU, hydroxyurea; MMS, methyl methane sulfonate; PAGE, polyacrylamide gel electrophoresis; hu-, human; ARS, autonomously replicating sequence(s); ASK, activator of S phase kinase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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